Biology 05LA – Fall Quarter 2013 Lab 3 – page 1

LAB 3: DIFFUSION, OSMOSIS AND THE PERMEABILITY OF CELL MEMBRANES

The movement of materials between cells and their external environment as well as within the cell is essential to life. For example, the raw materials for cellular metabolism must be imported and the metabolic waste products resulting from this metabolism must be exported. Within the cell, substrates for the cell’s enzymes must move into the active site of the enzyme and the soluble products of these reactions then distributed throughout the cell. Given the fundamental importance of this traffic, an understanding of the mechanisms whereby these materials move is essential.

Since both the internal and external cellular environments are aqueous, the materials to be moved are dissolved in water and thus form a solution (remember that in an aqueous solution, water is called the solvent and the materials dissolved in the water are called solutes). Solute movement may be separated into two broad categories. One of these requires the direct output of energy by the cell and is referred to as active transport. Here, a solute is moved against a concentration gradient of that solute, that is, from an area of low solute concentration to an area of high solute concentration. Thus active transport allows cells to generate concentration gradients that support a diverse array of cellular processes. You will be hearing much more about active transport in lecture. The other category of solute movement does not involve a direct expenditure of energy and is called diffusion. Diffusion will be the primary focus of today’s lab.

Your text defines diffusion as: “the spontaneous movement of a substance [solute] down its concentration gradient from a region where it is more concentrated to a region where it is less concentrated.” This movement continues until the solute is evenly distributed throughout the available space. The driving force for diffusion comes from the activity of the water (solvent) molecules in the solution. Water molecules are not static. In reality, they are in constant motion that is driven by the heat energy of the system. Evidence for this can be seen at high magnification in the light microscope when viewing suspensions of small particles rapidly vibrating in a random manner. This motion, referred to as Brownian motion, is the result of randomly directed collisions between the water molecules and the suspended particles. Similar interactions occur between solvent and solute molecules. The random nature of these collisions and the probability of more frequent collisions occurring in areas where solutes are more concentrated work together to move the solute molecules into the available space, that is, down their concentration gradient. Thus, anything that influences the number of collisions between solvent and solute can, in turn influence the rate of diffusion. Relevant possibilities here include: the initial concentration of the solute and the amount of thermal energy available (at higher temperatures the thermal agitation of the water molecules is more rapid and forceful). Diffusion rate is also influenced by the mass of the solute; more massive molecules have a greater inertia and thus require a greater force to move.

Thus far only the diffusion of solutes has been considered. Osmosis is an alternative version of diffusion that involves the movement of the solvent across a differentially permeable membrane separating two regions of different total solute concentration. A differentially permeable membrane is a barrier that allows the free passage of water molecules but does not allow the free passage of solute molecules. The region containing the lower total concentration of solutes is referred to as being hypotonic (relative to the other side) and the region containing the higher total solute concentration is referred to as being hypertonic (relative to the other side). Osmotic water movement occurs spontaneously from the hypotonic to the hypertonic side. Here, the driving force for osmotic water movement is the thermal motion of the water molecules as described above. The reason that movement is in the hypo- to hypertonic direction is that the total [chemical] potential energy of the

Biology 05LA – Fall Quarter 2013 Lab 3 – page 2

water molecules on the hypertonic side is diminished by the greater number of energy consuming interactions between solute and solvent on the hypertonic side. Thus, there is a potential energy gradient between the two sides; higher on the hypotonic side and lower on the hypertonic side. In this situation, water molecules will move down the energy gradient, that is, from the hypo- to the hypertonic side.

FACTORS INFLUENCING DIFFUSION RATE In these experiments we will study the influence of three factors upon diffusion rate. This will be done by making comparisons between the distances moved by colored solutes through a gelatin medium. The gelatin to be used is called agar and is composed of mostly water. As such, the agar approximates an aqueous system, but is much easier to use for this kind of study. The basic plan is that drops different solutions will be added to small test tubes containing agar. The tubes will then be incubated under the specified conditions for at least one hour. Since the incubation time for each of the tubes will be the same, the distances traveled by the solutes will be proportional to the relative diffusion rates of the solutes.

The solutes that will be used are: KMnO4 (potassium permanganate, mass = 158 grams/mole) and aniline blue (mass = 738 grams/mole). Table 1 lists the different solute concentrations and incubation temperatures that will be used. With some careful thought, you should be able to deduce which tube comparisons can be used to reveal the effects of varying molecular mass, solute concentration, and temperature upon diffusion rate.

Procedure:

1. Each group of 4 students should obtain 6 agar-filled test tubes and label them 1 – 6.

2. With Table 1 as your guide, and with the use of a different Pasteur pipette for each solute, add 1 drop of each of the specified solutions to the proper tube.

3. When all 6 tubes are complete, replace the stoppers and incubate each tube at the condition specified for that tube. Record the time.

4. After at least one hour, use a ruler to measure the diffusion distances of the solutes (see Fig. 1) and record these measurements in your lab notebook. Diffusion rate = distance moved (mm) / time (min.)

5. Remove the stoppers from the tubes and place them in the designated recycling container. Dispose of the tubes in the designated waste container.

Analysis. First, describe how these variations in molecular mass, solute concentration, and temperature influenced diffusion rate and then offer an explanation for these results based upon what you have learned about diffusion.

Tube # Solute Incubation

1 0.1 M KMnO4 4o C

2 0.1 M KMnO4 room temp.

3 0.1 M KMnO4 35o C

4 0.1 M An.Blue room temp.

5 0.02 M An.Blue room temp.

6 0.01 M An.Blue room temp.

Table 1.

clearagar

diffusiondistancesolute front

origin

falsesolute front

Figure 1. Measurement of diffusion distance.This diagram approximates the appearance of

a sample tube after the allotted incubation time. Diffusion distance will be the distance between

the origin at the top of the agar and the solute front. Ignore any false solute fronts that may appear

because the solute moved between the agar andthe wall of the tube.

Biology 05LA – Fall Quarter 2013 Lab 3 – page 3

OSMOSIS. Many cell types experience situations where the total solute concentration is different on the inside and the outside of the cell. When this occurs, water can move into or out of the cell depending upon the relative concentration of solutes on either side of the membrane. These water movements can have a significant influence upon cell volume or upon the level of hydrostatic pressure within the cell. In animals, osmotically driven water movement can drive many important secretory events such as sweating. However, in other animal cells, large changes in cell volume can be detrimental. In plants, osmotically driven water movement can contribute to a “skeletal” system that supports young plant parts or drive bulk solute flow through the plant. Given the diverse ways that these water movements influence both plants and animals, an understanding of the mechanism enabling this movement is essential to the biology student.

This study will also introduce another expression of solute concentration called osmolarity. As you should know, a 1 molar solution of a polar molecule like glucose contains 6.02 x 1023 molecules of glucose per liter of solution. However, when 1 mole of a salt like NaCl is combined with water to make a 1 molar solution, the NaCl dissociates to give 1 mole of sodium ions (Na+) and 1 mole of chloride ions (Cl-). The dissociation thus doubles the total solute concentration of this solution. Consequently there are twice as many solute molecules in this solution that can deplete the total potential energy of the water molecules in the solution. As a result, the osmotic effect of a 1.0 M NaCl solution is twice that of a 1.0 M solution of a polar molecule. Osmolarity is an expression of concentration that accommodates this situation. Here, what needs to be understood is that the osmolarity of a solution is an expression of the total concentration of solutes expressed as molarity. For example, a 1.0 M solution of NaCl has an osmolarity of 2 osmolar and a 1.0 M solution of CaCl2 has an osmolarity of 3 osmolar. Given this information, it follows that osmotic water movement will occur in response to differences in osmolarity but not necessarily to differences in molarity.

Modeling Osmosis: In this experiment, we will create “artificial cells” with the use of a synthetic differentially permeable membrane (called dialysis tubing). By filling these artificial cells (“dialysis bags”) with solutions of varying concentration and then placing them in beakers with solutions of varying concentration, we can model the osmotic challenges imposed upon real cells by changes in internal and external solute concentrations. Table 2 lists the 4 conditions that will be tested.

Constructing these dialysis bags is fairly simple. Dry, precut strips of dialysis tubing are soaked in a beaker of distilled water. The strip is removed from the water and one end of the strip is tied tightly with a short piece of string. The other end is then opened in a manner comparable to opening the plastic bags in the produce section of the supermarket. Once opened, the desired solution is put into the bag. The bag is then closed by tying a knot with one end of a longer piece of string. A piece of colored tape is attached to the free end of the string and marked with the appropriate number. The bag is then weighed and placed in the specified incubation solution. After one hour, the bags are reweighed to determine if water has moved into or out of the bag. In this manner should see how differences in solute concentration on either side of a membrane affect the direction of osmotic water movement.

Procedure:

1. Each table should obtain 2 – 400 ml beakers and then label each with either H2O or 0.5 M sucrose. The groups at each table will share these beakers.

2. Add 200 ml of the correct solution to the beakers.

Biology 05LA – Fall Quarter 2013 Lab 3 – page 4

3. Each lab group should obtain 4 strips of dialysis tubing and soak them in distilled water until they are soft and pliable.

4. With reference to Table 2, and using the method described above, create dialysis bags with the specified contents.

5. As each bag is finished, trim the excess string from each end of the bag (don’t cut off the label).

6. After weighing and recording that value (in grams), place them into the specified bathing solution.

7. Incubate the bags for about an hour (no longer). At the end of that time, quickly blot the excess solution from the surface of each bag, weigh it, and record the weight.

Analysis. For each trial, give the tonicity (either hypo-, iso-, or hypertonic) of the bag content relative to the bathing solution. After considering your results, what generalizations can you make about how differences in tonicity influence the direction of osmotic water movement?

Osmosis In Living Plant And Animal Cells. The osmotic relationships between plant and animal cells with their environment are closely regulated in both cell types, but are not identical. Much of this difference is related to some fundamental differences between these cells. Plant cells differ from animal cells in many ways, but only two are important to this discussion. First, plant cells possess a fairly rigid cell wall. Second, healthy plant cells are most often hypertonic in comparison to their immediate exterior. This circumstance has a significant osmotic consequence. Given the hypertonic condition of cell’s interior, water should move into the cells by osmosis. If the cells were not bounded by the cell wall, water would keep moving into the cell until it burst. This does not happen. Instead, the cell wall resists the predicted osmotic swelling and pressure builds up within the cell. Plants use this “turgor” pressure to provide support for the plant or to drive long distance translocation of solutes. A characteristic feature of most animal cells is that they usually lack any sort of a structure comparable to the plant cell wall. Thus, the cytoplasm of the animal cell must be maintained in osmotic balance with the cell’s exterior. That is to say that the osmolarity of the inside and the outside of the cell should the same (isotonic). Maintaining this osmotic relationship is vital to the health of the cells because an animal cell exposed to a hypotonic environment would rapidly swell and eventually burst. Conversely, an animal cell exposed to a hypertonic environment would shrink because of excessive water loss. The following exercises will demonstrate the behavior of plant and animal cells exposed to different osmotic environments.

In the first part of this exercise, we will manipulate the external solute concentration around the leaf cells of Elodea, and observe the consequences of this treatment with the light microscope. In the second part we will make similar observations on red blood cells (RBCs) exposed to hypo-. iso-, and hypertonic solutions. We will conclude by exposing rbcs to a range of glucose concentrations extending from hypo- to hypertonic in an attempt to estimate the osmolarity of the cells.

Bag # Bag Content Bathing Solutions

1 10.0 ml H2O water

2 10.0 ml H2O 0.5 M sucrose

3 10.0 ml 0.5 M

sucrose water

4 5.0 ml 0.5 M sucrose + 5.0 ml H2O

0.5 M sucrose

Table 2

Biology 05LA – Fall Quarter 2013 Lab 3 – page 5

Osmosis in the Leaf cells of Elodea. Elodea is a common aquatic plant used in many fresh water aquaria. Its leaves are thin and comprised of fairly large cells. These features allow the microscopist to make useful observations of cell structure in intact leaves.

Procedure:

1. With the use of a forceps, remove a healthy looking, green leaf from the tip of an Elodea twig. Make a wet mount of the leaf using water from the tank where it was collected. (Given this is fresh water, there are very few solutes dissolved in it. We will consider the osmolarity of this solution to be hypotonic relative to the leaf cells).

2. Examine the leaf first with low power (40x total magnification). Then select a suitable cell and focus on it with higher power (100x total magnification – no more!). Triangular cells that project from the edge of the leaf are most easily brought into clear focus, but if you focus carefully, cells the middle of the leaf can be studied.

3. Observe the following features in the healthy cell (it is important that you know what these cells look like in the healthy condition before applying the test solution):

• The cellulosic cell wall.

• The cell cytoplasm forms a layer just inside the cell wall.

• A large vacuole occupies the central core of the cell. This vacuole may be identified if you focus through the depth of the cell. It appears as a clear region in which no chloroplasts (globular green structures) come into clear focus.

• The chloroplasts are located in the thin layer of cytoplasm close to the cell wall. If the cell is healthy, the chloroplasts may be moving, but they always remain near the wall.

• The nucleus is often difficult to identify. It is found close to the cell wall and has a grey, shadowy appearance.

4. Once you have made the above observations, prepare another slide with a fairly large drop of 1.0 M NaCl. Then use a forceps to carefully transfer the Elodea leaf from your first slide to the drop of salt solution. Wait at least two minutes.

5. Apply a coverslip and blot off any excess salt solution. Observe the effect of this treatment upon the Elodea cells.

Analysis. Describe the structural change in the Elodea cells elicited by the NaCl treatment. Explain the structural change that you observed.

Osmosis in mammalian red blood cells. Red blood cells offer many advantages for this kind of study. They are readily available, easy to handle, and have a relatively simple structure characterized by a fixed size and unique shape (please see p. 911 of your text for a more detailed description of their structure). This latter feature will allow easy assessment of shape changes elicited by osmotic water movement. We will be using whole sheep blood obtained from a supply house. When handling these cells, you must keep in mind that you are dealing with a cell suspension. Thus the cells will tend to settle with time. As a result, you will need to resuspend the cells anytime they are transferred or observed.

At this point, your group should obtain 5 ml of the RBC suspension* for your experiments. Your TA will dispense this into a clean test tube which you take to your lab station and place into a small beaker that is partially embedded in ice – each table will have an ice bucket for this purpose.

[*Your TA will prepare this mixture by combining 6.0 ml of whole sheep blood with 40.0 ml of isotonic saline.]

Biology 05LA – Fall Quarter 2013 Lab 3 – page 6

Part 1: Correlation of RBC structure in response to exposure to solutes of different tonicity.

Procedure:

1. Obtain 3 small test tubes and label each with either hypo-, iso-, or hyper- (tonic).

2. In the proper tube, add 5.0 ml of hypo-, iso-, or hypertonic solution.

3. Once filled, add 0.2 ml of the RBC suspension to each of the 3 tubes -- keep in mind that you must re-suspend the RBCs by swirling the stock tube prior to dispensing.

4. Close each of these tubes with a small square of Parafilm and mix by gentle inversion.

5. Set these tubes aside for about 5 minutes before observation.

6. Students should now pair up to make the necessary microscopic observations.

7. For each of the samples, apply a drop of the RBC suspension to the center of a glass slide (remember to swirl before doing so), add a coverslip, and observe. [Caution, do not begin your observations with the hypotonic sample.] Do not dispose of your sample tubes at this time; you will need to record their appearance (either clear or cloudy) in your lab notebooks.

8. Make careful drawings in your lab notebook of a few of the RBC's from each of the samples. Note the shapes of the cells and their relative numbers for each of the samples. Record this information in your lab notebook along with the appearance of the tube (either clear or cloudy) from which they came.

Analysis. Describe the appearance the cells in each of the treatments. Explain the structural change that you observed.

Part 2: Estimation of the osmolarity of red blood cells.

An awareness of osmolarity is essential to biologists working with isolated cells and tissues because exposure of these isolates to anything but isotonic conditions would result in unintended swelling or shrinkage. Such changes would probably affect their experimental results. Thus, there must be some way to estimate the osmolarity of their experimental subjects. Actually there are several ways to do this, some requiring special instrumentation. We will use a relatively simple approach.

In this exercise, a fixed amount of the RBC suspension will be exposed to a series of solute concentrations extending from hypo- to hypertonic. Somewhere near the interface of these extremes, the cells will be isotonic with their environment. At this point, the osmolarity of the cells and their environment will be the same. That is, the rate of osmotic water movement into the cells will equal that moving out of the cells. Your task will be to approximate the location of this point. By now you should realize that the cells exposed to hypotonic conditions will swell and lyse (burst) and those exposed to hypertonic conditions will be intact. Thus, we will be seeking to identify the concentration change that results in a change from the lysed to the intact condition (this concentration should be close to isotonic).

Procedure: Two different solutes will be used for this study; glucose and NaCl. For each lab table, one group of 4 will use glucose and the other will use NaCl. Thus, each group will only need to prepare one set of dilutions as given in Table 3. When this study is complete, the two groups should share the data for the solute that they did not use.

1. Obtain 7 – 18x150 size test tubes and label each with one of the concentrations listed in the second column of Table 3.

Biology 05LA – Fall Quarter 2013 Lab 3 – page 7

2. Keeping in mind that you will be given a 0.5 M stock solution (C1) and that you will be making 10.0 ml (V2), of each concentration, you should now use C1V1 = C2V2 to solve for V1 (the volume of the 0.5M stock that will be needed to make the required dilution). Once calculated, record these values for V1 in the correct cells of Table 3.

3. Make the simple calculations needed to fill in column 4 of the table.

4. Make the required dilutions. Your TA will instruct you on how to do this efficiently.

5. Add 0.4 ml of the RBC suspension into each of the tubes (remember to re-suspend the cells before dispensing).

6. Close each tube with Parafilm, and mix by gentle inversion. Allow the samples to stand 5 minutes before collecting data.

Data collection. What you need to find here is the lowest solute concentration that contains intact cells.

In order to do this you will need to go back to the tubes made in Part 1 of this exercise and ask two questions: which of these tubes contain intact cells and is there any apparent difference between the tubes with intact cells verses those with lysed cells? You should see that there is an easy way to tell the difference.

Analysis. Using the visual feature alluded to above, what was the lowest glucose concentration at which intact cells were present? What was the lowest NaCl concentration at which intact cells were present? Explain this difference (remember that the values used are approximations).

Learning Goals/Desired Outcomes for Lab 3 1. Be able to define and differentiate between the following pairs or groups of terms:

a) solute - solvent. b) diffusion - osmosis. c) hypotonic - isotonic - hypertonic. d) molarity - osmolarity.

2. Be able to define the following terms: a) concentration gradient. b) differentially permeable membrane. c) tonicity.

3. Be able to explain how variations in solute molecular mass, solute concentration, and temperature influence diffusion rate.

4. For an aqueous system where two solutions are separated by a differentially permeable membrane, you should be able to predict the direction of osmosis given the molar and/or osmolar solute concentrations on both sides of the membrane.

5. You should be able to explain why osmolarity differences are more significant than molarity differences when trying to predict the direction of osmosis.

6. You should be able to predict how plant cells will respond to exposure to a hypertonic environment and to explain the reasoning behind your prediction.

7. You should be able to predict how animal cells will respond to exposure to hyper- and hypotonic conditions and to explain the reasoning behind your predictions.

8. For the exercise where you estimated the osmolarity of some red blood cells, you should be able to explain why the lowest solute concentration at which the cells were unbroken represented a reasonable estimate of the osmolarity of the cells.

TONICITY Desired molarity

(C2) V1 (ml)

ml H2O to be

added

(V2 - V1)

Hypotonic

Hypertonic

0.0

0.1

0.15

0.20

0.25

0.30

0.35

Table 3. Dilution Work Sheet

Biology 05LA – Fall Quarter 2013 Lab 3 – page 8

Biol 5LA Lab 3 In-class Worksheet Name __________________________

1. What is the driving force of diffusion?

2. How does the molecular weight of a solute affect the rate at which it diffuses in a solvent?

3. If a solution of CaCl2 has an osmolarity of 6, what is its molarity?

4. Why are osmolarity differences more significant than molarity differences when trying to predict the direction of osmosis?

5. In lab 1, why did you prepare the onion slide using water and the cheek cells using saliva? Use the terms you have learned in lab today in your answer.